1. Field of the Invention
The present invention relates to a reagent for analyzing solid components in urine and to a method for analyzing solid components in urine employing the reagent, and more particularly to a reagent employed for an optical analysis of solid components in urine by applying flow cytometry and a method for analyzing the same.
2. Related Art
In renal and urinary diseases such as infectious diseases, inflammatory lesions, degenerative lesion, calculosis, tumor and the like, various kinds of solid components appear in the urine depending on the disease. Examples of solid components include erythrocytes, leukocytes, epithelial cells, urinary casts, bacteria, fungi, crystals and mucus threads. Analyzing these components in urine is of great importance for early discovery of renal and urinary diseases and presumption of abnormal sites. For example, the measurement of erythrocytes is important in determining whether there is bleeding in the passageway from the nephric glomerulus to urethra. Appearance of leukocytes suggests suspicion about renal disease such as pyelonephritis, leading to early discovery of inflammation and infectious disease. By investigating urinary casts and morphological features of erythrocytes, the derivational sites thereof can also be inferred.
Conventionally, solid components in urine have been analyzed by visual microscopy using a microscope. This is performed by concentrating the urine to be tested by centrifugalization, then sometimes after staining the obtained sediments, putting them on a microscope slide so as to classify and count them under a microscope.
In recent years, an automatic measuring apparatus has been developed in which a flat sheath flow and an image processing technique are combined. The urine sample adjusted to flow in an extremely flat stream with a sheath liquid serving as an outer layer is filmed by a video recorder and the still picture thus obtained is subjected to image processing, thereby cutting out and displaying the images of solid components in the sample liquid. Observing the display, an examiner distinguishes and counts the solid components contained therein.
Further, for automatically classifying and counting the solid components in urine, Japanese Unexamined Patent Publication No. Hei 4(1992)-337459 discloses a reagent (employed) for an analysis of cells in urine by applying flow cytometry and a method for analyzing thereof. The reagent employed therein contains a fluorescent dye, an osmotic pressure compensating agent and a buffer agent. Various kinds of fluorescent dyes, osmotic pressure compensating agents and buffer agents are disclosed therein and a reagent employing Neutral Red or Auramine O as a fluorescent dye is described in an Embodiment.
Incidentally, it is desired to test the urine specimen as soon as possible after the specimen is taken out from the subject because the solid components degenerate and the number of bacteria increases in accordance with the passage of time.
Visual microscopy by microscope requires a lot of time and operation for the pretreatment of the urine specimen such as centrifugalization and concentration. Besides, microscopy is a great burden to the examiner. Also, the microscopy accuracy is low because the number of observed cells are small.
An automatic measuring apparatus using an image processing technique is advantageous to microscopy because the burden of microscopy is alleviated. However, when a lot of specimens are to be tested, it is not satisfactory enough because solid components need to be distinguished by an examiner and the processing speed is not speedy.
Moreover, distinguishing the solid components requires skill in both visual microscopy and examination by an automatic measuring apparatus using an image processing technique.
A method disclosed in Japanese Unexamined Patent Publication No. Hei 4(1992)-337459 wherein flow cytometry is applied to urine analysis have the advantage of quick measurement. However, on further investigation, the method was found to have the following problems.
(1) Appearance of crystals in the urine renders it difficult to distinguish between the crystals and the erythrocytes contained therein.
(2) When a lot of amorphous salts appear in the specimen, it becomes difficult to classify the other cells.
(3) In measuring a urine specimen containing hemoglobin or protein by using the reagent containing Auramine O having pH 8.5 shown in an Example of Japanese Unexamined Patent Publication No. Hei 4(1992)-337459, it may be impossible to measure the urine specimen accurately because of bonding the hemoglobin or the protein to the dye and depositing tiny sediments.
(4) Although the problem of (3) is solved by allowing the pH to be set at an acid value, stainability of the reagent declines at such pH value and, when yeast-like fungi appear in the specimen, it is difficult to distinguish erythrocytes from the fungi.
(5) When urine is analyzed by flow cytometry, it is necessary to repress the dilution ratio to be low because the quantity of solid components contained in the urine is small. However, when the dilution ratio is low and a fluorescent substance, for example, a pharmaceutical agent such as a vitamin or an antibiotic substance is excreted in the urine, it may be difficult to obtain sufficient fluorescent signal intensity of the solid components contained therein because the background fluorescence (background noise) of the urine itself is not negligible. When Neutral Red is used, there will be a great influence of the background fluorescence caused by the dye which does not bond to the cells.